The present invention relates to a system and a method for the continuous automated physical extraction of at least one liquid phase of a series of liquid microsamples which are taken beforehand as discrete packets in space and in time and are stored, and to an automated installation for carrying out, in succession and continuously, the taking of such microsamples, in storing them and in extracting at least one phase thereof and optionally measurements thereof. The invention applies more particularly, but not exclusively, to whole blood microsamples taken from a mammal, such as a rat or a mouse.
It is known to use centrifugal force to separate the various phases or components of liquid microsamples, such as whole blood from mammals, in biology, for the purpose of physically extracting, by centrifuging, at least one phase of these microsamples. However, the high speed of rotation that characterizes current centrifuges prevents them in practice from operating when their charge is not balanced, i.e. when all the microsamples are not placed at locations intended to accommodate them during this rotation, which typically consist of microcontainers housed in the centrifuge.
In the case of whole blood microsamples, various techniques are used to collect, filter and then separate the whole blood into constituents of different densities. It is general practice to use a micropipette and microcontainers that may already be in place in the centrifuge and that are filled in turn until the complete filling capacity of the centrifuge has been reached. The latter is then made to undergo rapid rotation, for a suitable time, so that the various components of the blood (such as serum/plasma on the one hand, and red corpuscles on the other) are separated inside each microcontainer.
For example, mention may be made of document US-A-2004/0166551 which has a centrifuge in which the microcontainers are holders each having an upper portion for being filled with the microsample, said portion being extended, via a narrowing in its cross section, by a lower portion for separating the constituents of this microsample, such as blood.
It is also known to use an extraction agent facilitating this separation within total blood microsamples, the density of which is intermediate between that of the various phases of the blood and which consists of a polymer gel. For example, mention may be made of document U.S. Pat. No. 5,906,744 for the use of such an extraction gel, which is by nature thixotropic.
When blood microsamples are sequentially transferred to such microcontainers for the centrifuge, there arises, apart from the problem of the uncertainty in the volume precision due to the micropipette used for the transfer, the aforementioned problem, which lies in the need to fill all of the microcontainers with microsamples in order to carry out the centrifugation, thereby preventing the microsamples from being centrifuged progressively as they are transferred into these microcontainers owing to the imbalance (i.e. the lack of balancing of the load in the centrifuge). In general, the smallest filling volume used in a centrifuge accommodating these microcontainers is about 200 μl.
Another major drawback of known centrifugation extraction systems and methods for blood microsamples is that an operator is normally required for sequencing the operations of taking these microsamples from the mammal in question and then temporarily storing them, followed by the operations of transferring the microsamples taken and stored into the microcontainers of the centrifuge in order to carry out the centrifugation.
The main automated systems known for taking blood microsamples derive from the field of molecular imaging in preclinical research on small mammals. In particular, to measure the concentration of an endogenous molecule, an imaging technique known as positron emission tomography (PET for short) is used, via the pharmacokinetic modeling of a radiotracer administered intravenously. In practice, the radiotracer concentrations are measured by this technique over the course of time at various points in the organism, called “organs”, and in the compartment that delivers the radiotracer to all the organs, that is to say the arterial blood. The tomographs for small animals are used to extend the field of PET to preclinical trials in rodents, and to make diagnostic and therapeutic research benefit from this technique. On the down side, it becomes difficult to measure the arterial fraction by the usual blood sampling methods. This is why micromethods have been developed for measuring PET radiotracers in small animals, especially rats and mice.
A first micromethod of measurement was developed by a group of Canadian research workers at Sherbrooke University, for the purpose of continuous automatic sampling. In small animals (rats and mice), this group thus developed a system (see the article by Convert et al., IEEE Transactions on Nuclear Science, Vol. 54, No. 1, February 2007, 173) commercialized by the company AMI (Advanced Molecular Imaging)/Gamma Medica. To summarize, in this system the blood is continuously extracted from the animal using a pull-syringe but no collecting of samples is possible. This has the drawback of not meeting the requirements of new tracers that require the subsequent recovery of blood samples.
It may be noted that the same group had developed in 1998 (see the article by D. Lapointe et al. “A Microvolumetric Blood Counter/Sampler for Metabolic PET Studies in Small Animals”, IEEE TRANSACTIONS ON NUCLEAR SCIENCE, VOL. 45, No. 4, AUGUST 1998) a system for extracting 10 μl blood samples and taking them automatically into a positron-sensitive counter. The computer-controlled blood sampler was based on a segmentation of the blood microsamples by air microbubbles (1 to 3 μl). At the end of the experiment, the tube thus formed could be cut, thus making the microsamples available for subsequent biochemical analysis. The treatment of these specimens has the drawback of not being able to be carried out automatically on the sampling line.
A second measurement micromethod was developed by a group of American researchers (Department of Molecular and Medical Pharmacology, UCLA School of Medicine & Department of Molecular and Medical Pharmacology) and relates to the automated taking of samples and their collection. The reader may usefully refer to the article by Huong-Dun Lin et al., Automatic Control System of a Microfluidic Blood Sampler for Quantitative microPET Studies in Small Laboratory Animals, 2006 IEEE Nuclear Science Symposium Conference Record), which describes a blood sampling system on a microfluidic chip that collects blood samples of small volume (180 nl) with an adjustable sampling time. In this system, the minimum time between two samples is 2 seconds and the amount of liquid deposited depends on the precision of the micropipette. In this microfluidic chip, only 18 blood samples could be collected.
The preliminary results have allowed blood kinetics to be derived with the “FDG” radiotracer on mice. Since the total blood loss is less than 3.5 μl, the impact on the physiological change is reduced to a minimum. On the down side, it is necessary to wait until the sampling procedure has been completed for the blood samples to be rinsed off the chip and into microtubes before then being counted in a gamma detector of the “well” type. Although this constraint is not very detrimental at the start of an entry function, in which the first samples are taken very close together, the same does not apply in the case of the last samples which are much more spaced apart and for which the time interval continues to increase. Thus, the gamma counting of the first of the 18 samples is carried out a very long time after it was taken. Since in addition the activity to be measured itself decreases strongly, the signal-to-noise ratio becomes very degraded.
Other drawbacks of this second method lie in the relatively long minimum sampling time between samples, being 2 seconds, and by the fact that the radiotracer counting on whole blood is carried out only at the end of a sequence (a minimum of 1 h after injection), which makes this method difficult to implement for tracers such as 11C which has a half-life of 20 minutes (there remains only one-eighth of the amount of tracer after 1 h, since this is equivalent to three half-lives). Furthermore, this rapid decrease in the quantity to be measured is considerably accentuated by the difficulty in separating the plasma (containing the radiotracer) from the corpuscles. Specifically, for sample volumes of 0.18 μl sufficient separation for carrying out differentiated measurements in line may even be considered to be impossible.